RNA-polypeptide interactions are essential for fundamental cellular and viral activities such as transcription and translation. Accordingly, significant effort has been expended to develop methods of identifying polypeptide factors that interact with RNA. Convenient and sensitive systems to effectively identify polypeptide factors that interact with RNA are being sought. One approach has focused on affinity purification of polypeptides using RNA sequences fused to affinity labels or tags. Affinity labels are typically able to bind to cognate ligand molecules thus providing a means for separating the tagged RNA molecule from a mixture.
For example, Hunt et al., Genes and Dev. 13:437-438 (1999) and others have described a procedure for covalently linking RNA to cyanogen bromide activated sepharose through a free amine group available on the RNA. Polypeptides that bound to the RNA sequence were identified by exposing the sepharose-linked RNA to cellular extract (see, e.g., Brown et al., J. Biol. Chem. 275 (10): 7424-72429 (2000); Kaminski et al., RNA 1:924-938 (1995). In a related method, Walter et al., RNA 5:1570-1585 (1999) described the use of agarose-adipic acid hydrazide to immobilize oxidized RNA and identify RNA binding proteins.
Lee et al., Nucleic Acids Res. 30(2): 429-438 (2002) and others (see, e.g., Rodgers et al., Anal. Biochem. 277(2): 254-259 (2000); Mehta et al., Mol. and Cell. Biol. 18(8): 4426-4432(1998)) have identified RNA binding polypeptides using a biotin linked RNA and affinity purification using streptavidin columns.
Finally, Bachler et al., RNA 5(11): 1509-1516 (1999) have identified RNA binding polypeptides using an heterologous RNA fused to a RNA aptamer sequence that binds to streptomycin, which is immobilized on sepharose beads (see also WO 01/48480).
There is a need, however, to further develop a novel, convenient and sensitive systems to effectively identify polypeptide factors that interact with RNA.